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Test ID DHRF Dihydrorhodamine Flow Cytometric N-Formyl-Methionyl-Leucyl-Phenylalanine Test, Blood

Useful For

Evaluation of RAC2 deficiency and RAC2 gain of function

Reporting Name

DHR Flow fMLP, B

Specimen Type

WB Sodium Heparin

Shipping Instructions

Specimens are required to be received in the laboratory weekdays and by 4 p.m. on Friday. Collect and package specimen as close to shipping time as possible. Ship specimen overnight in an Ambient Shipping Box-Critical Specimens Only (T668) following the instructions in the mailer.


It is recommended that specimens arrive within 24 hours of collection.


Samples arriving on the weekend and observed holidays may be canceled.

Necessary Information

Ordering physician name and phone number are required.

Specimen Required

Both a whole blood sodium heparin specimen and a whole blood sodium heparin control specimen from an unrelated, healthy donor are required.


Supplies: Ambient Shipping Box-Critical Specimens Only (T668)



Container/Tube: Green top (sodium heparin)

Specimen Volume: 5 mL

Collection Instructions: Send whole blood specimen in original tube. Do not aliquot.


Normal Control:

Container/Tube: Green top (sodium heparin)

Specimen Volume: 5 mL

Collection Instructions:

1. Collect a control specimen from a normal (healthy), unrelated person within an hour of the patient's specimen collection time.

2. Label clearly on outermost label normal control.

3. Send whole blood specimen in original tube. Do not aliquot.

Specimen Minimum Volume

1 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
WB Sodium Heparin Ambient 48 hours GREEN TOP/HEP

Reject Due To

Gross hemolysis Reject
Gross lipemia Reject

Clinical Information

This assay can be used for the diagnostic evaluation of RAC2 deficiency, which is a neutrophil defect that causes profound neutrophil dysfunction with decreased chemotaxis, polarization, superoxide anion production, azurophilic granule secretion. This disease is caused by inhibitory variants in the RAC2 gene, which encodes a Rho family GTPase essential to neutrophil activation and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function.(1) Patients with RAC2 deficiency have been shown to have normal neutrophil oxidative burst when stimulated with phorbol myristate acetate (PMA), indicating normal NADPH oxidase activity, but abnormal neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP), which is a physiological activator of neutrophils. The defective oxidative burst to fMLP, but not to PMA, is consistent with RAC2 deficiency.(2) By contrast, gain of function variants in RAC2 would lead to a an exaggerated response to fMLP.(3)

Reference Values

Result name


Cutoff for defining normal

% fMLP ox-DHR+






Control % fMLP ox-DHR+



Control MFI fMLP ox-DHR+




fMLP = N-formyl-methionyl-leucyl-phenylalanine

DHR = dihydrorhodamine

MFI = mean fluorescence intensity


The appropriate age-related reference values for Absolute Neutrophil Count will be provided on the report.


An interpretive report will be provided, in addition to the quantitative values described in Clinical Information.


Interpretation of the results of the quantitative dihydrorhodamine (DHR) flow cytometric assay has to include both the proportion of positive neutrophils for DHR after N-formyl-methionyl-leucyl-phenylalanine stimulation, and the mean fluorescence intensity.


Specimens are optimally tested within 24 hours of blood draw, though the stability of the assay is within 48 hours of collection. Specimens should be collected in sodium heparin and transported under strict ambient conditions. Use of the Ambient Mailer-Critical Specimens Only box (T668) is encouraged to ensure appropriate transportation of the specimen.


Hemolyzed specimens may give high background. Specimens with an absolute neutrophil count below 200 will not be accepted for this assay. Complete myeloperoxidase deficiency can yield a false-positive result.

Supportive Data

Dihydrorhodamine analysis was performed to assess neutrophil oxidative burst in 157 healthy donors, 74 children, and 83 adults.

Clinical Reference

1. Ambruso DR, Knall C, Abell AN, et al: Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation. Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4654-4659

2. Accetta D, Syverson G, Bonacci B, et al: Human phagocyte defect caused by a RAC2 mutation detected by means of neonatal screening for T cell lymphopenia. J Allergy Clin Immunol. 2011 Feb;127(2):535-538

3. Hsu AP, Donko A, Arrington ME, et al: Dominant activating RAC2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects. Blood. 2019 May 2;133(18):1977-1988

Method Description

A sodium heparin whole blood specimen is incubated at 37° C in the presence of DHR123. N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulant is added and mixed with the whole blood specimen for additional incubation at 37° C. The sample is then centrifuged, and cell pellet is subsequently lysed with ammonium chloride at room temperature. Lysed samples are then washed with azide-free phosphate buffered saline prior to staining with LIVE/DEAD viability marker and CD15 at ambient temperature. Finally, cells are washed, centrifuged, and resuspended in 1% paraformaldehyde prior to analysis. Viable neutrophils are identified by the use of the viability dye and further confirmed by the presence of CD15. Approximately 20,000 viable neutrophil events in the unstimulated sample are used to set the limits for number of events collected for flow cytometry. The results are derived as delta % DHR+ neutrophils after fMLP stimulation and mean fluorescence intensity.(O'Gorman MR, Corrochano V: Rapid whole-blood flow cytometry assay for diagnosis of chronic granulomatous disease. Clin Diagn Lab Immunol 1995;2[2]:227-232; Kuhns DB: Diagnostic testing for chronic granulomatous disease. Methods Mol Biol. 2019;1982:543-571)

Report Available

3 to 4 days

Test Classification

This test was developed using an analyte specific reagent. Its performance characteristics were determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information


LOINC Code Information

Test ID Test Order Name Order LOINC Value
DHRF DHR Flow fMLP, B 98123-3


Result ID Test Result Name Result LOINC Value
ANC Absolute Neutrophil Count 751-8
FMPPP % FMLP ox-DHR+ 85373-9
FMPM MFI fMLP ox-DHR+ 85370-5
ANCC Control Absolute Neutrophil Count 85369-7
FMPPC Control % fMLP ox-DHR+ 85372-1
FMPMC Control MFI fMLP ox-DHR+ 85371-3
DHRFI Interpretation 69052-9

Method Name

Flow Cytometry